1. Field of the Invention
The invention relates to a method of protein purification, particularly to a method for purifying protein by chromatography.
2. Description of the Prior Arts
Ion exchange chromatography is one of the most common procedures for protein purification. Conventional ion exchange chromatography includes loading a protein sample in a direction to a column packed with ion exchanger to allow charged groups on proteins to interact with immobilized groups on the ion exchanger, whereby proteins can be absorbed by the ion exchanger, which is usually described as an equilibrium model (Giddings, J. C., Unified Separation Science, Wiley-Interscience, New York, 1991. pp. 16-36). Subsequently, a gradient of salt solution is introduced into the column from the same direction as the protein sample, whereby proteins binding to the ion exchanger to different extents are respectively eluted at different salt concentrations. However, ion exchange chromatography lacks a specific affinity. Therefore, separating a target protein from undesired impurity by conventional ion exchange chromatography usually requires further purification, such as affinity chromatography or preparation of specific antibodies, which dramatically elevates cost of protein purification.
In analytical chemistry, a method called “back flush” has been developed to better separate inorganic ionic species on ion exchangers. By simply reversing the flow direction of elution relative to that of sample loading, this method is aimed to minimize peak spreading (Haddad et al., J. Chromatogr., 1985, 318: 279-288), avoid problems of irreversible ion exchange (Roberts et al., Anal. Chem., 1981, 53: 1691-1695) and reduce the dip peak (Okada, T. and Kuwamoto, T., J. Chrormatogr., 1985, 350: 317-323). Specifically, by loading the sample into the column in the opposite direction to that in which the target ion is eluted, the band broadening is minimized in the concentrator column through compression of the sample ions into a compact band during the initial stages of elution from the concentrator column (Haddad et al., supra). Because the direction of process can affect the result of separation, not only equilibrium interactions but also nonequilibrium interactions may play a role in this method.
Nevertheless, contrary to simple inorganic ions, proteins are large molecules consisting of multiple amino acids. Current techniques for purifying proteins suffer from band broadening and usually require further purification from elution fractions from a broad salt concentration range to obtain a homogenous protein. In the other words, there is still a need for obtaining a protein with higher homogeneity.
To overcome the shortcomings, the present invention provides a method for purifying protein to mitigate or obviate the aforementioned problems.